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Biomedical subjects

A Goldfarb

Publications and source records attributed to A Goldfarb.

At least 91 records · Page 5Linked to original sources
PubMed1991

Mapping of a contact for the RNA 3' terminus in the largest subunit of RNA polymerase.

Stalled elongation complexes of Escherichia coli RNA polymerase were prepared carrying the photo-cross-linkable 8-azido derivative of adenine at the 3'-terminus of the nascent RNA chain. Ultraviolet irradiation of such complexes resulted in the cross-linking of radiolabeled RNA exclusively to the beta' subunit of RNA polymerase. The adduct was mapped between Met932 and Trp1020 in the linear sequence of the beta' polypeptide using specific chemical degradation of the cross-linked species.

Adenosine Monophosphate↗
PubMed1991

Mapping of trypsin cleavage and antibody-binding sites and delineation of a dispensable domain in the beta subunit of Escherichia coli RNA polymerase.

We have mapped principal sites in the Escherichia coli RNA polymerase molecule that are exposed to attack by trypsin under limited proteolysis conditions. The 1342-amino acid-long beta subunit is alternatively cleaved at Arg903 or Lys909. The cleavage occurs adjacent to a dispensable domain (residues 940-1040) that is absent in the homologous RNA polymerase subunits from chloroplasts, eukaryotes, and archaebacteria. In E. coli, this region can be disrupted with genetic deletions and insertions without the loss of RNA polymerase function. Insertion of 127 amino acids into this region introduces a new highly labile site for trypsin proteolysis. The dispensable domain carries the epitope for monoclonal antibody PYN-6 (near residue 1000), which can be used for anchoring the catalytically active enzyme on a solid support. We also report the identification of a secondary trypsin cleavage at Arg81 of the beta' subunit within a putative zinc-binding domain that is conserved in prokaryotes and chloroplasts.

Amino Acid Sequence↗
PubMed1991

Mapping of the priming substrate contacts in the active center of Escherichia coli RNA polymerase.

The active center of DNA-dependent RNA polymerase performs the principal biochemical reaction of gene expression. Using cross-linkable substrate analogs and site-directed mutations, two evolutionarily invariant amino acids in the beta subunit of the Escherichia coli enzyme (Lys1065 and His1237) were mapped close to the binding site of the priming substrate of the reaction. Surprisingly, the mutational substitution of these residues (Lys1065----Arg and His1237----Ala) did not inactivate the catalytic function, but inhibited transition from the initiation to the elongation stage of transcription.

Affinity Labels↗
PubMed1991

Development of a bone marrow culture for maintenance and growth of normal human B cell precursors.

The absence of long term bone marrow cultures for studying the growth and differentiation of human B cell precursors (BCP) has placed restrictions on the ability to analyze the early stages of human B cell ontogeny. We now describe a bone marrow-derived adherent cell microenvironment that maintains human BCP for several weeks in vitro. The adherent cells are maintained in a serum-free tissue culture medium, and consist of a predominant population of CD10+ fibroblast-like cells and a minor population of CD10+/nonspecific esterase+ macrophages. Adherent cell cultures seeded with fresh or cryopreserved fetal bone marrow, or purified CD10+/surface IgM- cells, provide a supportive microenvironment for lymphoid cells with a predominant phenotype of CD10+/CD19+/HLA-DR+/surface IgM-. Supplementation of the adherent cell cultures with human IL-7 induces active growth of BCP during the first 14 to 21 days of culture. However, the expansion of these cells does not continue past 21 days, and the cultures undergo a steady decline in BCP. Analysis of adherent cell conditioned medium revealed the presence of an unidentified soluble factor (or factors) that acts in concert with IL-7 to promote the growth of CD10+/surface IgM- cells. This culture system will be useful in elucidating the patterns of gene expression and growth factor requirements that characterize normal human B cell ontogeny, and perturbations of normal B cell ontogeny that lead to immunodeficiency and leukemia.

Antigens, CD↗
PubMed1991

lac repressor acts by modifying the initial transcribing complex so that it cannot leave the promoter.

RNA polymerase engaged in the joint complex with the lac repressor at the lac UV5 promoter cannot escape into elongation but generates abortive RNA oligomers. The joint complex actively transcribes a few initial base pairs in a reaction unusually sensitive to a decrease in the substrate concentration. The joint complex, however, fails to traverse a point in the initial transcribed sequence that normally requires a high concentration of the elongating substrate. Thus, the repressor acts by augmenting a natural high "kNTP" site (pause site) embedded in the promoter. A lethal RNA polymerase mutation that mimics the effect of the repressor leads to an analogous block of promoter clearance and shortened abortive product pattern on several promoters, reflecting the widespread occurrence of high kNTP sites in promoters.

Base Sequence↗
PubMed1991

A beta subunit mutation disrupting the catalytic function of Escherichia coli RNA polymerase.

The substitution of the evolutionarily conserved Glu-813 for lysine in the beta subunit of RNA polymerase (RNAP) causes a partial loss of function in the assembled RNAP. In the presence of the four ribonucleoside triphosphates, the mutant RNAP displayed a decreased frequency of promoter clearance and diminished elongation rate. Both defects could be compensated by raising the ribonucleoside triphosphate concentration. In the abortive initiation reaction limited by the incomplete set of ribonucleoside triphosphates, the mutant RNAP generated aberrant patterns of products indicative of their enhanced loss from the RNAP-promoter complex. A model is proposed, attributing the multiple effect of the mutation to the malfunctioning of the RNAP active center.

Amino Acid Sequence↗
PubMed1991

In vivo platelet activation in beta-thalassemia major reflected by increased platelet-thromboxane urinary metabolites.

Increased frequency of thromboembolic events has been recently observed in patients with beta-thalassemia major (TM). Platelet function anomalies including impaired aggregation, increased circulating aggregates, and our finding of shortened platelet survival indicate that platelets may be involved in the hypercoagulability in thalassemia. Consequently, we used a technique based on thin layer chromatography purification and enzyme immunoassay to measure urinary metabolites of thromboxane A2 (TXA2) and prostacyclin (PGI2) in nine splenectomized patients with beta-TM regularly transfused, five non-splenectomized patients with beta-thalassemia intermedia (TI), and 20 healthy individuals. A significant 4- to 10-fold increase was observed in the urinary excretion of 2,3-dinor-TXB2, 11-dehydro-TXB2 and 2,3-dinor-6-keto-PGF1 alpha in patients with TM and TI as compared with healthy controls. No significant differences were found in the concentrations of these metabolites between TM and TI patients. Six TM patients received a very low dose of aspirin (20 mg/day) for 7 days. A significant decrease was observed in the urinary concentrations of 2,3-dinor-TXB2 and 11-dehydro-TXB2 derived from platelets. However, the levels of urinary 2,3-dinor-6-keto-PGF1 alpha reflecting vascular production and TXB2 and 6-keto-PGF1 alpha originating from the kidney were not significantly changed. These results are consistent with those of increased in vivo production of TXA2 because of endogenous platelet activation.

6-Ketoprostaglandin F1 alpha↗
PubMed1991

Comparison of cardiac catheterization and Doppler echocardiography in the decision to operate in aortic and mitral valve disease.

Clinical decisions utilizing either Doppler echocardiographic or cardiac catheterization data were compared in adult patients with isolated or combined aortic and mitral valve disease. A clinical decision to operate, not operate or remain uncertain was made by experienced cardiologists given either Doppler echocardiographic or cardiac catheterization data. A prospective evaluation was performed on 189 consecutive patients (mean age 67 years) with valvular heart disease who were being considered for surgical treatment on the basis of clinical information. All patients underwent cardiac catheterization and detailed Doppler echocardiographic examination. Three sets of two cardiologist decision makers who did not know patient identity were given clinical information in combination with either Doppler echocardiographic or cardiac catheterization data. The combination of Doppler echocardiographic and clinical data was considered inadequate for clinical decision making in 21% of patients with aortic and 5% of patients with mitral valve disease. The combination of cardiac catheterization and clinical data was considered inadequate in 2% of patients with aortic and 2% of patients with mitral valve disease. Among the remaining patients, the cardiologists using echocardiographic or angiographic data were in agreement on the decision to operate or not operate in 113 (76% overall). When the data were analyzed by specific valve lesion, decisions based on Doppler echocardiography or catheterization were in agreement in 92%, 90%, 83% and 69%, respectively, of patients with aortic regurgitation, mitral stenosis, aortic stenosis and mitral regurgitation. Differences in cardiac output determination, estimation of valvular regurgitation and information concerning coronary anatomy were the main reasons for different clinical management decisions. These results suggest that for most adult patients with aortic or mitral valve disease, alone or in combination, Doppler echocardiographic data enable the clinician to make the same decision reached with catheterization data.

Adult↗
PubMed1991

Trichomegaly, pigmentary degeneration of the retina and growth disturbances. A probable autosomal recessive disorder.

Two brothers are described with trichomegaly, early pigmentary degeneration of the retina, growth retardation, anterior pituitary deficiencies and peripheral neuropathy. This syndrome, initially reported in a boy by Olivers and Mac Farlane in 1965 (6), and thereafter in six sporadic cases of both sexes, is not associated with a recognizable chromosomal defect. The present report of two brothers of healthy parents with negative familial history suggests an autosomal recessive mode of inheritance of this entity.

Adult↗
PubMed1990

Blocking of the initiation-to-elongation transition by a transdominant RNA polymerase mutation.

RNA polymerase, the principal enzyme of gene expression, possesses structural features conserved in evolution. A substitution of an evolutionarily invariant amino acid (Lys1065----Arg) in the beta subunit of Escherichia coli RNA polymerase apparently disrupts its catalytic center. The mutant protein inhibited cell growth when expressed from an inducible promoter. The assembled holoenzyme carrying the mutant subunit formed stable promoter complexes that continuously synthesized promoter-specific dinucleotides but that did not enter the elongation step. The mutant polymerase inhibited transcription by blocking the access of the wild-type enzyme to promoters.

Amino Acid Sequence↗
PubMed1990

Recombinant RNA polymerase: inducible overexpression, purification and assembly of Escherichia coli rpo gene products.

The genes, rpoA, rpoB and rpoC of Escherichia coli, which encode the RNA polymerase alpha-, beta- and beta'-subunits, respectively, have been individually placed on expression plasmids under the control of the bacteriophage T7 promoter. Induction of the T7 RNA polymerase gene in host cells harboring each of the three plasmids resulted in the extensive overproduction of the three polypeptides. The overproduced subunits were purified and assembled into a functional enzyme, whose specific activity and dependence on the sigma-factor were indistinguishable from native RNA polymerase purified by conventional methods.

Cloning, Molecular↗
PubMed1990

Myopathological findings in thalassemia major.

In thalassemia major (TM) one third of patients suffers from muscle wasting, weakness and cramps. Six patients with TM were studied. All had muscle wasting and proximal weakness; serum levels of vitamin E were low (0.6-7.0 micrograms/dl) while CPK, LDH and aldolase were normal. EMG revealed low-amplitude short-duration polyphasic potentials in 3 patients and normal activity in 3 others. Nerve conduction velocities were normal in 3 patients studied. On muscle biopsies, moderate variation in fiber size with fiber atrophy and preponderance of type 1 fibers were discovered. Our findings confirm the existence of nonspecific myopathic changes in TM. Chronic vitamin E deficiency should be considered in the pathogenesis of the myopathy in TM.

Adolescent↗
PubMed1990

High incidence of cholelithiasis in older patients with homozygous beta-thalassemia.

The records of 48 patients with homozygous beta-thalassemia were reviewed for evidence of cholelithiasis by abdominal ultrasonography or plain abdominal X-ray. The presence of cholelithiasis was reported in 25 patients (52%). The incidence increased with age to 83% in patients over 31 years of age. Gallstones were more frequent among patients with beta-thalassemia intermedia and with less blood transfusion requirements. Eleven patients with cholelithiasis (44%) became symptomatic. Eight received operations on an elective or semielective basis. Another patient had incidental cholecystectomy during splenectomy. The preoperative evaluation included abdominal ultrasonography, nucleotide biliary scan and evaluation of the cardiopulmonary status. Due to the longer survival of patients with homozygous beta-thalassemia, the problem of cholelithiasis is becoming more frequent.

Adolescent↗
PubMed1990

Cardiopulmonary assessment in beta-thalassemia major.

Thalassemia patients succumb at a young age to congestive heart failure. Hitherto, attention has been focused on left ventricular function. This report emphasizes right ventricular dysfunction and abnormal pulmonary function. We performed cardiopulmonary evaluation, including echo-Doppler, spirometry, CO diffusion (DCO), and blood gas analyses in 35 patients with homozygous beta-thalassemia maintained by multiple blood transfusions. Six autopsy lung specimens were studied. Thalassemia patients exhibited pulmonary dysfunction, characterized by hypoxemia (85 percent of the patients were outside the 95 percent confidence limits), reduced lung volumes (51 percent), flow rates (63 percent) and DCO (50 percent). Right ventricular dysfunction was more prevalent than left ventricular dysfunction. Furthermore, 75 percent of the patients had evidence of pulmonary hypertension consistent with more frequent right ventricular rather than left ventricular dysfunction. Our findings suggest that in thalassemia patients, complex cardiopulmonary abnormalities precede the final outcome of congestive heart failure.

Adult↗
PubMed1989

Platelet survival in patients with beta-thalassemia.

Thromboembolic events associated with significant morbidity and mortality have been observed in patients with beta-thalassemia major (TM). These include arterial as well as venous thrombosis and the development of early arteriosclerosis. To elucidate the possibility that TM patients may develop a hypercoagulable state we carried out a study of platelet kinetics on ten patients with TM and four patients with thalassemia intermedia (TI). Autologous platelets were labeled with indium-111-oxine, and the platelet lifespan (PLS) was determined. A significant shortening of PLS was observed in 13 out of 14 patients examined. The mean PLS (+/- 1 SD) in ten patients (8 TM, 2 TI) who underwent splenectomy was 107 +/- 36 hr (control splenectomized 248 + 51 hr) (P less than .001) and in four nonsplenectomized patients (2 TM, 2 TI) was 102 +/- 64 hr (control 224 + 23 hr) (P less than .01). The short PLS in addition to reported findings of increased circulating platelet aggregates and the decreased response of TM platelets to aggregating agents suggests in vivo platelet activation in thalassemic patients.

Adolescent↗
PubMed1989

Expression of cloned rpoB gene of Escherichia coli: a genetic system for the isolation of dominant negative mutations and overproduction of defective beta subunit of RNA polymerase.

The rifampin resistance rifD18 allele of rpoB, carried on the expression plasmid pXT7 beta, is controlled by a strong bacteriophage T7 late promoter and two weak Escherichia coli promoters. Depending on the host strain, pXT7 beta specifies different levels of Rifr beta subunit, providing a system for the isolation, maintenance, and overexpression of dominant lethal alleles of rpoB. In rpoB+ hosts, pXT7 beta confers the Rifr phenotype on the Rifs host. Negative rpoB mutations in the plasmid DNA can thus be scored by screening transformants for Rifs. In an rpoB(Am) supD(Ts) host in which chromosomal rpoB expression is decreased as the temperature goes up, some of the negative plasmid-borne rpoB mutations displayed a dominant phenotype. In a host harboring inducible T7 RNA polymerase, the defective beta subunits could be overexpressed independently of the E. coli transcriptional machinery. With this system, we isolated several negative rpoB mutations induced in vitro by hydroxylamine. Seven of the mutant rpoB alleles, when overexpressed, were found to specify normal-size beta polypeptides. Two of them displayed the dominant lethal phenotype in the rpoB(Am) supD(Ts) background. We also constructed a mutation (rpoB1800) in which 24 carboxy-terminal amino acids were substituted with a random 19-amino-acid sequence. The nonfunctional rpoB1800 beta polypeptide was isolated and assembled in vitro into the core enzyme molecule.

Cloning, Molecular↗